This invention relates to a method for attaching nucleic acids to surfaces to permit structural analysis of the nucleic acids. More particularly, this invention relates to a method for attaching DNA or RNA to a gold surface for structural analysis of the DNA or RNA by Auger electron spectroscopy, angle-dependent x-ray photoelectron spectroscopy, scanning tunneling microscopy, atomic force microscopy, and the like.
Structural analysis of nucleic acids suddenly became important in 1953 when Watson and Crick discovered that deoxyribonucleic acid (DNA) is the biological molecule that stores genetic information and transfers that information from generation to generation. In succeeding years, ribonucleic acids (RNAs) were also shown to play a role in genetics as messenger RNAs and transfer RNAs. Further, studies of viruses such as tobacco mosaic virus (TMV) demonstrated that RNA was capable of serving as a repository of genetic information.
Prior to 1977, structural analysis of DNA and RNA was difficult, expensive, and time-consuming. However, in that year two methods for determining the sequence of bases in DNA were discovered independently that made nucleotide sequencing of DNA easier, cheaper, and faster. These two methods are the chemical degradation method of Maxam and Gilbert, 74 Proc. Nat'l Acad. Sci. USA 560-64 (1977), and the chain termination method of Sanger, 74 Proc. Nat'l Acad. Sci. USA 5463-67 (1977). At about the same time, techniques of molecular cloning were developed so that DNA copies of RNA molecules could be cloned and sequenced by these two methods. Direct RNA sequencing techniques were also developed.
The chemical degradation method of DNA sequencing is based on the concept of partially degrading DNA fragments through four base-specific degradation reactions, one for each of the four bases. Degradation of a base makes the phosphodiester backbone more susceptible to chemical cleavage. Thus, after the bases are specifically degraded, the DNA fragments are subjected to another reaction to break the phosphodiester backbone. The specifically degraded fragments are then size fractionated. Labeling of the fragments with radioactive, fluorescent, chemiluminescent or other labels permits the fragments to be detected. Thus, a nucleotide can be identified and assigned to each position in the nucleotide chain by the reaction that specifically degraded the base at that position.
The chain termination method also relies on four different reactions to deduce the identity of each nucleotide in a chain. However, these reactions involve synthesis of nucleotide chains rather than their degradation. DNA is normally double-stranded and each base in a nucleotide chain is bonded to a complementary base in the other nucleotide chain. Guanine (G) pairs with cytosine (C) and adenine (A) pairs with thymine (T). Thus, if one knows the sequence of one strand of the DNA, the sequence of the other strand is also known. DNA polymerases are available which will synthesize a complementary strand of DNA if provided with a single-stranded DNA template and an oligo- or polynucleotide primer for providing a hydroxyl group to which the next nucleotide in the chain is attached. Addition of chain terminating nucleotide analogs, such as 2',3'-dideoxynucleoside triphosphates that lack the hydroxyl group to which the next nucleotide in the chain would ordinarily be attached, makes it possible to terminate a chain at every possible nucleotide position. By using four chain-terminating reactions, each one, respectively, containing a chain terminating analog of one of the four nucleotides in DNA, the sequence of nucleotides in the chain can be determined. The chains are labeled as in the chemical degradation technique with radioactive, fluorescent, chemiluminescent, or other labels. The chains are then fractionated by length. In this way the sequence of nucleotides in the chain can be deduced by identifying the nucleotide analog that terminates a chain at each position in the DNA.
Despite the huge improvement that these two techniques have been to determining the structure of DNA molecules at the nucleotide sequence level, significant additional improvements are still needed to increase the speed and reduce the cost of sequencing large nucleic acids. Without such improvements it will not be feasible to sequence the entire three billion basepairs of DNA that comprise the entire genetic complement of a human being in a timely and economical manner. Other large sequencing projects will, likewise, be impractical. Alternative methods of nucleotide sequencing to those just described are being developed.
A method that has been suggested for rapid sequencing of nucleic acids is through imaging of the nucleic acid with techniques such as scanning tunneling microscopy (STM) and atomic force microscopy (AFM). These recently developed methods utilize microscopes that make it possible to resolve or visualize individual atoms in some samples. In principle, it should be possible to attach nucleic acids to suitable substrates and visualize the nucleic acids by STM or AFM. The nucleotide sequence of a nucleic acid could be read by visually identifying each base from an image of the nucleic acid, or by detecting some other non-visual signal (spectroscopic) which is unique to the various bases. In practice, the potential for sequencing nucleic acids by STM or AFM imaging has suffered from several technical stumbling blocks. For example, suitable substrates are needed that are both atomically flat and free of contaminants or other artifacts that would interfere with producing readable images. A further problem has been that the nucleic acid must adhere to the substrate so that the nucleic acid does not move during the minutes that are needed to produce the image. Clearly, a moving target is not an ideal subject for a readable image. Thus, a method of attaching nucleic acids to an atomically flat, contaminant free substrate so that the nucleic acids are firmly anchored and do not move would be very important to structural analysis of nucleic acids using methods that distinguish the atomic or molecular structure of the nucleic acid. Further, a method is needed for binding the nucleic acid to the substrate so that the nucleic acid is oriented for imaging of the bases. Binding the phosphodiester backbone of the nucleic acid to the substrate would, thus, seem to be the best way of attachment for these purposes.
Binnig and Rohrer, the inventors of STM, were the first to image DNA molecules, G. Binnig et al., 49 Phys. Rev. Lett. 57-60; G. Binnig & H. Rohrer in Trends in Physics at 38-46, J. Janta & J. Pantoflicek, eds. (European Physical Society, The Hague, 1984), however progress has gone only as far as resolving the major and minor grooves of uncoated DNA, T. Beebe et al., 243 Science 370-72 (1989); G. Lee et al., 244 Science 475-77 (1989), and distinguishing purines from pyrimidines in uncoated polynucleotides, D. Dunlap and C. Bustamante, 342 Nature 204-06 (1989). These experiments were conducted with highly oriented pyrolytic graphite (HOPG) substrates. HOPG was originally the obvious substrate of choice because of its advantages of having limited reactivity, low surface roughness (less than 5 .ANG. vertical deviation over hundreds of .ANG. lateral distance), reproducibility of flatness, low cost, and ease of preparation. However, many ambiguous HOPG surface structures could be confused with deposited biomolecules, C. Clemmer & T. Beebe, 251 Science 640 (1991), thus making HOPG an undesirable surface for this work. Thus, other substrates were sought.
Prior to 1983, gold had not been extensively investigated as a substrate for chemisorption studies due to its relative inertness towards molecular oxygen, and even carbon monoxide interacted only weakly. Nuzzo and Allara, 105 J. Am. Chem. Soc. 4481-83 (1983), discovered that thiols and disulfides could be adsorbed from solution to form ordered monolayers on gold films. The monolayers are formed because of relatively strong (30-40 kcal/mole) covalent bonds between the sulfur and gold molecules.
Bain et al., 111 J. Am. Chem. Soc. 321 (1989), and Bain et al., 111 J. Am. Chem. Soc. 7155 (1989), described further the nature of the bonds formed between organosulfur compounds and gold. The following information is extracted from these two articles. There is a specific interaction of gold with sulfur and other "soft" nucleophiles and a low reactivity toward most "hard" acids and bases. The strong specific interaction between gold and sulfur atoms in thiols, disulfides, and certain other sulfur-containing compounds induces spontaneous assembly of an adsorbed monolayer at the gold-solution interface. It is the formation of strong, coordinative gold-sulfur bonds that drives the spontaneous assembly of these monolayers. It is the position of Bain et al. that the species ultimately formed on the gold surface by adsorption of thiols from solution is a thiolate (Au-SR). It is stated, however, that the mechanism by which an initially physisorbed thiol is converted to a chemisorbed thiolate remains unclear. In other words, while the mechanics of the chemistry are not clear, the fact that bonds are formed is known. Monolayers of alkanethiols on gold appear to be stable indefinitely in air or in contact with liquid water or ethanol at room temperature.
Concerning the kinetics of formation of monolayers, Bain et al., 111 J. Am. Chem. Soc. 321, 328 (1989), stated that the rate of formation of a self-assembled monolayer is influenced by many factors, some of which can be controlled relatively easily, such as temperature, solvent, concentration and chain length of the adsorbate, and cleanliness of the substrate. Other factors, such as the rate of reaction with the surface and the reversibility of adsorption of the components of the monolayer, are inherent to the system. They concluded that experimental conditions must be established for each new system studied. At moderate concentrations (ca. 1 mM), the adsorption process is characterized by two distinct phases, an initial period of rapid adsorption lasting a few minutes in which the monolayer reaches a thickness of 80-90% of its maximum, and a slower period lasting several hours, during which the thickness slowly approaches its final value. This behavior can be rationalized by rapid adsorption of an imperfect monolayer followed by a slower process of additional adsorption and consolidation, possibly involving displacement of contaminants, expulsion of included solvent from the monolayer, and lateral diffusion on the surface to reduce defects and enhancing packing.
This discovery led to work involving a wide range of applications in fields including electrochemistry, biology, and microlithography. Related chemical systems have been utilized for STM of DNA molecules. In L. Bottomley et al., 10 J. Vac. Sci. & Tech. 591 (1992), a gold surface was activated by reaction with N,N-dimethyl-2-mercaptoethylamine to create a monolayer of exposed cationic groups. DNA was then bound to the monolayer by coulostatic interactions. This result offered one possible resolution to the major problem of holding nucleic acids in place during STM and AFM imaging.
U.S. Pat. No. 5,106,729 to Lindsay et al. describes a method of attaching base-substituted, phosphate-substituted, or sugar-substituted polynucleotides to gold substrates for analysis by STM or AFM. In this method, oxygen atoms in the base, phosphate, or sugar of the polynucleotide are replaced by sulfur atoms. The sulfur-containing polynucleotide is then treated with a mercury compound or other metal-containing compound to form complexes between the mercury or other metal and the sulfur atoms for enhancing contrast during the imaging process. Then, the metal-complexed polynucleotide is attached to the gold substrate by Faradaic deposition (electrodeposition) by holding the gold substrate about 1-2 V positive with respect to a reference electrode. It is believed that the mercury forms an amalgam with the gold substrate, thus binding the polynucleotide to the substrate. Subsequent imaging is performed in water. This method does not ensure that covalent bonds are formed directly between the nucleic acid and the substrate to firmly attach the nucleic acid to the substrate nor that the bases are exposed and unreacted so that they can be imaged or otherwise analyzed.
Herein is described a method of using gold-thiol monolayer chemistry to anchor nucleic acids for imaging by STM and AFM. Instead of activating the gold substrate for binding of the nucleic acids by coulostatic interactions, or binding the nucleic acids to the gold substrate by forming an amalgam between a nucleic acid-metal complex and the gold substrate, the nucleic acids are activated to permit covalent bonding of the phosphate backbone of the nucleic acid to the gold substrate, thus leaving the bases exposed and unreacted for imaging and analysis. In view of the foregoing discussion, it will be appreciated that these advantages are a significant advancement in the art.